During the last funding period we found that the major enteric neurotransmitters regulate the open probability of CA2+ channels and Ca2+ influx indirectly, by controlling membrane potential. In general excitatory transmitters cause depolarization by activation of non- selective cation currents and inhibitory transmitters activate K+ conductances to cause hyperpolarization and reduced excitability. We will investigate several novel K+ conductances to cause hyperpolarization and reduced excitability. We will investigate several novel K+ conductances that are involved in setting membrane potential and regulation rhythmicity. We have found recently that several families of inward rectifier conductances (Kir) are expressed by colonic muscles, and we will investigate how each class of these channels contributes to electrical activity. Ba2+ - sensitive current (possibly due to expression of Kir2.1 channels) appear to contribute to membrane potential. Kir 3 genes are also expressed, and the conductance resulting from expression of members of this family could help sustain the phasic nature of electrical activity during responses to excitatory agonists. Kir6 transcripts have also been identified, and these channels, in combination with sulfonylurea receptor protein subunits (SUR 2B) may form K/ATP channels in colonic muscles. The resulting conductance appears to contribute to resting membrane potential and may be activated further by agonists. We will also study how small conductance Ca/2+ - activated K+ channels are regulated and participate in enteric inhibitory neural responses, and we will investigate how rapidly inactivating, voltage-dependent K+ conductances affect smooth muscle excitability and responses to pacemaker activity. We will also study how Ca2+-activated Cl- currents are activated and contribute to responses to neurotransmitters. We have recently found evidence for expression of a stretch-activated Cl- conductance in colonic muscles. Currents resulting from this conductance will be characterized, and we will attempt to determine how this conductance contributes to the response of intact muscle to stretch. Finally, we will study the effects of phosphorylation on Ca2+ currents in colonic muscles and determine how the mechanisms by which this conductance is directly regulated by neurotransmitters and hormones. These studies will greatly expand our understanding of the basic ion mechanisms that set membrane potential and regulate excitability in colonic muscles.